Free sulfurous acid and bound sulfurous acid have been widely used as food addivites for bleaching or preserving food (e.g., antioxidant) in food processing. However, it has been suggested that sulfurous acids might cause certain allergies and have harmful effects on health if the concentration of the sulfurous acids is excessive in food. In order to prevent the adverse effects of sulfurous acids on health, Food Sanitation law provides strict guidelines on the sulfurous acid content in each food item. The food industry must not only comply with the law but also take great caution regarding the sulfurous acid content in food where bound sulfurous acids are added.
Several methods have been utilized for measuring the free and bound sulfurous acid content. For example, Monier Williams method, distillation-iodometry method, distillation-colorimetry method (these methods are described in Inspection Guidelines I for Food Sanitation, 1973, Nippon Food Sanitation Association ed., p 404-408) and Rankine method (Rankine, B.C., 1962, Aust. Wine Brewing Spirit Rev 80, 14) are typically used to measure the amount of bound and free sulfurous acids in food. These methods are fairly complicated, even for skilled workers, and requires an extra step (e.g., distillation), a large amount of samples and a long period of time for analysis. Another problem with these methods is that the sulfurous acid to be measured is difficult to recover because the sulfurous acid evaporates from the system during distillation or remains as residue in the system. Alternatively, there are enzymatic methods for measuring the sulfurous acid content such as sulfuric acid oxidase recovered from the chicken liver (Beutler, H. O., 1984, Food Chem 15: 157) or instruments such as ion chromatography (Kim, H. I., Kim, Y. K., 1986, I. Food. Sci. 51 1360), gas chromatography (Hamano, H., 1978, I. Food Sanitation 19 '56), polarography (Holak, W., Specchio, I., 1989, I. Assoc. Off. Anal. Chem. 72: 476). The enzymatic method does not provide reliable results because the presence of colored substances or ascorbic acid in a sample to be tested interferes with the accurate quantitation of sulfurous acid. In addition, the enzyme itself is not stable in the sample. The methods using the instruments described above are not desirable either because the instruments are expensive or because of the drawback noted regarding the enzymatic method (e.g., interference of colored substance or ascorbic acid).
We have previously disclosed the method of measuring sulfurous acid using an enzyme recovered from a bacterium belonging to the genus Thiobacillus (Nakamura, K., Amano, Y., Nakayama, O., 1989, Appl. Microbiol. Biotechnol. 31: 351). The enzymatic method is preferable for measuring the sulfurous acid content in a sample due to the following features: specificity of the enzyme contained in the bacterium to sulfurous acid (a property which gives accurate measurement), stability of the enzyme in the bacterial cell, and ease of manipulation. The enzymatic method, however, cannot be used to measure the total sulfurous acid content (free sulfurous acid+bound sulfurous acid compounds) of the sample. This prior method is only useful for measuring the free sulfurous acid content in a sample. Therefore, a method of measuring the total sulfurous acid content in a sample is needed.